The antibody-drug conjugate (ADC) field has made significant advances with the FDA approval of Brentuximab Vedotin for the treatment of a select group of patients and with the advancement of many other ADCs in the clinic. The linker component of ADCs is one important feature in developing optimized therapeutic agents that are highly active at well tolerated doses. The electrophilic maleimide functional group has proven very useful in the preparation of ADCs due to its high degree of specificity for reacting with thiol groups and the very fast thiol addition kinetics under gentle conditions.

As has been noted by multiple investigators in the bioconjugate field, the thio-substituted product of the reaction between the electrophilic maleimide functional group and free thiol of antibody is subject to slow elimination, thus reversing the above reaction:

When this reversible reaction occurs in a purified preparation of the ADC, the reaction is largely undetectable because the maleimide and thiol which are regenerated through the elimination process simply react again, thus reforming the intact conjugate. However, when other thiols are present, the net effect can be the transfer of the maleimide from the antibody of the ADC onto any other available thiol. This process has been documented to occur in plasma, in which the maleimide of an ADC transfers to cysteine 34 of serum albumin (Alley et al., Bioconjugate Chem. 2008, 19, 759-765). This process has also been reported when an ADC is incubated in the presence of excess cysteine or glutathione (Jununtula et al., Nature Biotech, 2012). The present invention provides, inter alia, bioconjugates that do not undergo this transfer reaction.